Mitochondria are essential, complex organelles of eucaryotic organisms required for a variety of metabolic processes including the generation of energy by oxidative phosphorylation. In humans the critical importance of maintenance of mitochondrial function is underlined by the occurrence of severe disease caused by a variety of mutations in mtDNA. The biogenesis and maintenance of mitochondrial function requires the function of molecular chaperones such as Hsp70. Our long term goal is to understand the mechanism of action of molecular chaperones within mitochondria using Saccharomyces cerevisiae as a model system. Using genetic and biochemical approaches we will analyze the roles of mitochondrial chaperones of the Hsp70 class in the processes of protein translocation, folding and assembly of mitochondrial proteins and maintenance of mitochondrial DNA. The Hsp70 of the mitochondrial matrix, Ssc1, is an essential component of the apparatus required for translocation of proteins from the cytosol. Ssc1 is tethered to the import channel via its interaction with an essential peripheral component of the channel, Tim44.Mge1, an essential nucleotide release factor for Ssc1 is also a component of this complex. How this complex functions in cycles of binding and release of the translocating polypeptide is not understoood. Mdj1, a DnaJ-related protein of the matrix, although apparently not involved in the translocation process, facilitates folding of imported proteins. Focusing on a genetic approach involving the isolation and analysis of suppressor mutations and temperature-sensitive alleles, the functional interrelationships of these chaperone components of the mitochondrial matrix will be dissected. Ssh1 and Ssj1 are recently-identified Hsp70s of the mitochondrial matrix. To understand the functional relationship among the mitochondrial Hsp70s, Ssc1, Ssh1 and Ssj1, a combination of biochemical and genetic approaches will be pursued. For example, the biochemical properties of these 3 Hsp70s will be compared. Extragenic revertants of the cold-sensitive phenotype of ssh1 cells will be studied to determine the function of this Hsp70 in mitochondria.